Expression of protein-coding genes is a multi-step process beginning with transcription by RNA polymerase II in the nucleus. During transcription, the nascent pre-mRNA undergoes several processing steps including capping, splicing, and polyadenylation. The mature mRNA is then exported to the cytoplasm for translation. Although each of the steps in gene expression is carried out by a distinct cellular machine, growing evidence indicates that there is an extensive network of coupled interactions between each machine. At present, however, little is understood about the mechanisms involved in the coupling. The goal of this proposal is to determine the mechanisms for coupling transcription, splicing, and mRNA export. In Specific Aim 1, a functional assay will be used to determine whether transcription by RNA polymerase II promotes mRNA export. In addition, the recently identified TREX complex, which may function in coupling all three processes, will be investigated in detail. All of the full-length human TREX components will be cloned, expressed, and antibodies generated. These reagents will be used to test the function of the TREX complex and to determine how this complex associates with active genes and their transcripts. In Specific Aim 2, the mechanism for coupling transcription to splicing will be investigated using an in vitro system for coupling transcription by RNA polymerase II to splicing. Studies will also be carried out to determine whether splicing factors, including U1 and U2 snRNPs, are recruited to actively transcribed genes. Finally, in Specific Aim 3, coupling of splicing to mRNA export will be investigated by isolating and determining the composition of the spliced mRNP that promotes export. In addition, the role of the conserved DEAD box helicase protein UAP56, which functions in export and is also present in both the TREX complex and spliceosome, will be determined using specific mutations in this protein combined with functional assays for coupling.